3D printing microneedle as well as preparation method and application thereof

Abstract

The invention relates to a 3D printing antibacterial microneedle, the microneedle is of a multilayer bionic mushroom-shaped structure made of a drug-loading needle tip layer, a needle body layer, a drug-loading storage layer and a substrate layer, and the volume ratio of the drug-loading needle tip layer to the needle body layer to the drug-loading storage layer to the substrate layer is 1: 1: (20-50): (20-50); the drug-loaded needle tip layer is prepared by loading a nano-composite on soluble polyvinylpyrrolidone; the needle body layer is prepared by concentrating methylacryloylated polylysine and methylacryloylated sodium hyaluronate and then carrying out photo-co-crosslinking on the concentrated methylacryloylated polylysine and methylacryloylated sodium hyaluronate; the medicine carrying storage layer is prepared by adopting soluble polyvinylpyrrolidone to load a nano complex; the substrate layer is prepared after non-degradable polyethylene glycol diacrylate is subjected to photo-crosslinking. The micro-needle is integrally prepared by combining 3D printing with a polydimethylsiloxane mold turning method, the micro-needle has excellent mechanical performance and puncture performance, autonomous drug delivery at a wound part is facilitated, accurate release and efficient uptake of a drug and a cascade effect of'tissue permeation-bacterial targeting-gene silencing 'are achieved, and the micro-needle has a good application prospect. The wound healing time is remarkably shortened, the targeted delivery efficiency, bioavailability and safety effectiveness of antibacterial drugs are remarkably improved, and the problem that nucleic acid drugs are difficult to be used for resisting wound infection is solved.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a 3D printed microneedle, a preparation method thereof and an application thereof. Background Art

[0002] With the popularization of high-energy damage weapons, the pattern of war injuries has undergone a fundamental change, and the incidence of complex skin injuries in war wounded has increased sharply. Full-thickness skin defects provide an ideal physiological environment for pathogen colonization, resulting in skin traumatic infections and chronic non-healing wounds, and even leading to amputation and death. The interaction between "environment-microorganism-host" significantly changes the wound microecology under severe environmental fluctuations, and accelerates the biofilm formation rate of bacteria such as Staphylococcus aureus (about 2.3 times higher). The complex battlefield environment poses stringent requirements on the stability of the antibacterial drug delivery system. First, dust particles may block about 30% of the microchannels; second, the harsh battlefield environment requires the wounded to have a high level of self-help and mutual rescue capabilities and drugs that are convenient for self-administration; third, the rapid evolution of drug-resistant bacteria, the physical barrier of biofilms and the extreme battlefield environment pose severe challenges to antibiotic therapy.

[0003] Wound healing mainly includes four stages of hemostasis, inflammation, proliferation and tissue remodeling, which are cascaded and partially overlapped. Among the pathogens isolated from skin wound pus specimens, Gram-positive bacteria (GPB) are mainly Staphylococcus aureus, Staphylococcus haemolyticus and Enterococcus faecalis; Gram-negative bacteria are mainly Escherichia coli, Pseudomonas aeruginosa and Proteus. In the treatment of wound infections, injection administration is likely to cause systemic exposure risks, while cream agents have defects such as low tissue permeability, which restricts the local treatment effect of antisense oligonucleotides. CRISPR-Cas gene editing, engineered phages and antimicrobial peptides are used as new antibacterial drugs, but their translational applications are limited due to potential risks such as immunogenicity and off-target effects.

[0004] Antisense oligonucleotides (ASOs) recognize and bind to specific mRNA sequences through the Watson-Crick base pairing rule. They have high specificity of action, low risk to human gene expression, are easy to design and synthesize, can target any possible gene, and are used to control the spread of drug-resistant microorganisms, antibacterial and anti-infection, etc. Compared with other therapies, antisense oligonucleotide therapy technology has the following advantages: First, antisense oligonucleotides have high specificity. Antisense oligonucleotides can precisely regulate the expression of bacterial growth metabolism, virulence factors and drug-resistant genes by specifically binding to the target mRNA through sequence specificity. Without affecting other genes, it specifically regulates the expression of target genes, enabling antisense oligonucleotides to effectively target and inhibit the expression of drug-resistant genes when treating bacterial infections and thus overcoming the problem of antibiotic resistance; Second, the design and synthesis of antisense oligonucleotides are relatively simple and flexible. The design of antisense oligonucleotides mainly depends on the sequence information of the target mRNA, and antisense oligonucleotides targeting different bacterial virulence factors and drug-resistant genes can be rapidly developed. For example, modification methods such as phosphorothioate (PS) and peptide nucleic acids (PNA) can significantly enhance the stability and biological activity of antisense oligonucleotides; Third, the diverse mechanisms of action of antisense oligonucleotides in cells. Antisense oligonucleotides inhibit gene expression through RNase H-mediated mRNA degradation, and can also achieve gene silencing by blocking mRNA translation. The efficient uptake and effective release of antisense oligonucleotides are two prerequisites for achieving antisense oligonucleotide-mediated bacterial gene silencing.

[0005] The delivery of antisense oligonucleotides is limited by problems such as delivery vectors, bacterial penetrability, and electrostatic repulsion of the bacterial cell wall, resulting in a low conversion efficiency of antisense oligonucleotides in bacteria and difficulty in meeting clinical needs. The peptidoglycan layer of Gram-positive bacteria, which is 30-100 nm thick, forms a molecular sieve with a pore size of <2 nm, and the negatively charged teichoic acid strongly repels antisense oligonucleotides; the outer membrane porins of peptidoglycan only allow hydrophilic molecules with a molecular weight of <600 Da to passively diffuse, while the molecular weight of antisense oligonucleotides is usually >2000 Da. Studies have shown that the effective delivery rate of unmodified antisense oligonucleotides in bacteria is less than 0.3%, and 92% are actively excreted by efflux pumps. Existing studies have shown that the bacterial ATP-binding cassette (ABC) transporter pathway can specifically uptake glucose polymers (GPs) with a poly[4-O-(α-D-glucopyranosyl-α-D-glucopyranoside)] structure, such as maltodextrin, etc., and use bacterial-specific ABC transporters to transport small molecule antibiotic conjugates modified with specific sugar skeletons and PNA-modified nanospheres into bacteria.

[0006] At present, the clinical regimen for antisense oligonucleotide drugs requires repeated administration of higher therapeutic doses (high dose-high frequency administration) to maintain effective drug concentrations, which increases the cost of treatment and is more likely to cause dose-related toxic side effects. In clinical treatment, antisense oligonucleotides are required to penetrate the biological barriers of target organs or target tissues to enhance enrichment in the lesion area. However, due to the influence of cell wall charge, the delivery efficiency of negatively charged DNA still needs to be improved. In addition to the obstruction of the bacterial membrane wall structure, the skin, scabs and granulation tissues all hinder the penetration of antibacterial drugs, and there are problems such as low delivery efficiency, safety and effectiveness to be improved, and high delivery costs. To this end, it is necessary to develop a drug delivery technology that can efficiently deliver and effectively release antisense nucleotide drugs to meet the clinical needs of effectively treating trauma and skin traumatic infections.

[0007] Microneedles have a microstructure with a high aspect ratio and puncture ability. They are painless, minimally invasive, highly effective, minimally invasive, can directional penetrate the stratum corneum and physiological barriers, and increase the contact area and depth with the wound surface. As a new transdermal drug delivery technology, they are used for localized drug delivery to the skin and wounds. However, the use of microneedles to combat complex wound infections faces many challenges. First, wound exudate leads to insufficient adhesion of microneedles, which easily fall off and fail. Second, the drug loading capacity of a single-layer microneedle is limited, making it difficult to achieve long-term sustained release and antibacterial effects. Third, microneedle preparation processes (such as molding) have problems such as low precision and poor yield when manufacturing complex biomimetic structures, which seriously restricts their clinical application.

[0008] 3D printing (3DP) is an additive manufacturing and rapid prototyping technology. It builds objects by printing materials layer by layer from a three-dimensional Computer Aided Design (CAD) model. This technology includes powder binding (PB), fused deposition modeling (FDM), semi-solid extrusion (SSE), and stereolithography (SLA). By selecting printing materials, optimizing process parameters, and designing model structures (such as size, shape, structure, and dosage), 3DP allows for precise control of drug appearance, dosage, and release characteristics, providing more precise, effective, and personalized therapeutics. Therefore, the development of a novel multifunctional in situ microneedle-based delivery system (MNDS) is crucial to address the treatment of complex wound infections. Summary of the Invention

[0009] The object of the present invention is to provide a 3D printed antibacterial microneedle, and the microneedle is a bionic mushroom-like structure made of a drug-loaded tip layer, a needle body layer, a drug-loaded reservoir layer and a base layer. Among them, the volume ratio of the drug-loaded tip layer: the needle body layer: the drug-loaded reservoir layer: the base layer is 1:1:20-50:20-50;

[0010] The drug-loaded tip layer is prepared by loading a nano-complex (ASO@GP-SiNPs) with soluble polyvinylpyrrolidone (PVP). The mass-volume ratio of soluble polyvinylpyrrolidone to the nano-complex solution in the drug-loaded tip layer is 100-200:1 (mg / mL);

[0011] The needle body layer is prepared by photo-crosslinking after concentrating poly-L-lysine methacryloyl (PLMA) and hyaluronic acid methacrylate (HAMA). The mass ratio of poly-L-lysine methacryloyl to hyaluronic acid methacrylate in the needle body layer is 1:0.25-1;

[0012] The drug-loaded reservoir layer is prepared by loading a nano-complex with soluble polyvinylpyrrolidone. The mass-volume ratio of soluble polyvinylpyrrolidone to the nano-complex solution in the drug-loaded reservoir layer is 100-200:1 (mg / mL);

[0013] The base layer is made by photo-crosslinking of non-degradable polyethylene glycol diacrylate (PEGDA).

[0014] In a preferred technical solution of the present invention, the volume ratio of the drug-loaded tip layer: the needle body layer: the drug-loaded reservoir layer: the base layer is 1:1:30:50.

[0015] In a preferred technical solution of the present invention, the mass-volume ratio of soluble polyvinylpyrrolidone to the nano-complex solution in the drug-loaded tip layer is 150:1 (mg / mL).

[0016] In a preferred technical solution of the present invention, the mass ratio of poly-L-lysine methacryloyl to hyaluronic acid methacrylate in the needle body layer is 1:0.5-1.

[0017] In a preferred technical solution of the present invention, the mass-volume ratio of soluble polyvinylpyrrolidone to the nano-complex solution in the drug-loaded reservoir layer is 150:1 (mg / mL).

[0018] In a preferred technical solution of the present invention, the drug-loaded reservoir layer is a circular patch.

[0019] In a preferred technical solution of the present invention, the base layer is a square patch.

[0020] In a preferred technical solution of the present invention, the nano-complex is made of antisense oligonucleotides (ASOs): dextran nanosilica particles (GP-SiNPs) in a mass ratio of 1:100 - 300. Among them, the antisense oligonucleotides are selected from any one or a combination of nucleotide sequences against Gram-positive bacteria and nucleotide sequences against Gram-negative bacteria. The dextran nanosilica particles are made by covalently binding dextran (GP) to the surface of nanosilica particles (SiNPs). The dextran is selected from any one or a combination of maltodextrin (MD) and diethylaminoethyl dextran (DEAE).

[0021] In a preferred technical solution of the present invention, the antisense oligonucleotides are composed of a nucleotide sequence against Gram-positive bacteria and a nucleotide sequence against Gram-negative bacteria.

[0022] In a preferred technical solution of the present invention, the nucleotide sequence against Gram-negative bacteria is SEQ ID NO.1.

[0023] In a preferred technical solution of the present invention, the nucleotide sequence against Gram-positive bacteria is SEQ ID NO.2.

[0024] In a preferred technical solution of the present invention, the antisense oligonucleotides are made of nucleotide sequence SEQ ID NO.1: nucleotide sequence SEQ ID NO.2 in a mass ratio of 1:2 - 2:1, preferably 1:1.

[0025] In a preferred technical solution of the present invention, the dextran is made of maltodextrin: diethylaminoethyl dextran in a mass ratio of 0.5 - 2:1, preferably 1 - 1.5:1.

[0026] In a preferred technical solution of the present invention, the hydrodynamic diameter of the nano-complex is 50 - 300 nm, preferably 80 - 200 nm.

[0027] In a preferred technical solution of the present invention, the diameter of the nanosilica particles is 2 - 3 nm.

[0028] In a preferred technical solution of the present invention, the hydrodynamic diameter (Dynamic Light Scattering, DLS) of the dextran nanosilica particles is 5 - 8 nm.

[0029] In a preferred technical solution of the present invention, the encapsulation efficiency of the nano-complex for the antisense oligonucleotides is ≥85%, preferably ≥90%, more preferably ≥95%.

[0030] In the preferred technical solution of the present invention, the uptake rate of any one of the anti-Gram-positive bacteria and anti-Gram-negative bacteria to the nano-complex is ≥30%, preferably ≥35%, and more preferably ≥40%.

[0031] In the preferred technical solution of the present invention, the polyvinylpyrrolidone is selected from any one of PVP K30, PVP K60, PVP K90 or a combination thereof.

[0032] In the preferred technical solution of the present invention, the concentration of the polyvinylpyrrolidone is 10-30%, preferably 13-18%.

[0033] In the preferred technical solution of the present invention, the preparation of the nano-complex comprises the following steps: Under the condition of stirring (1000-2000 rpm), the antisense oligonucleotide: dextran nanosilica particles are placed in a mass ratio of 1:100-300 at 2°C - 8°C and shaken and mixed for 1-2 h, centrifuged at (5000-10000) g * (15-30) min, and eluted 3-5 times to obtain the nano-complex.

[0034] In the preferred technical solution of the present invention, the preparation temperature of the nano-complex is 4°C - 10°C.

[0035] In the preferred technical solution of the present invention, the preparation of the dextran nanosilica particles comprises the following steps:

[0036] (1) Mix maltodextrin: diethylaminoethyl dextran in a mass ratio of 0.5-2:1, and add water to make a dextran solution with a concentration of 10-20 mg / mL;

[0037] (2) Under the condition of stirring (100-200 rpm), place the dextran solution and the required amount of nanosilica particle solution at 50-100°C for reaction for 3-6 h, then add the required amount of NaBH4 to the reaction solution and react for 8-12 h, and then ultrafilter and centrifuge the reaction solution 4-6 times to obtain the dextran nanosilica particles.

[0038] In the preferred technical solution of the present invention, the centrifugation condition is (7500-9000 rpm) * (15-20 min).

[0039] In the preferred technical solution of the present invention, the mass-to-volume ratio of NaBH4: reaction solution is 1:10-20, preferably 1:15-16.

[0040] In the preferred technical solution of the present invention, the nanosilica particles are prepared by ultraviolet irradiation of a precursor obtained by mixing a silicon oxygen coupling agent and 1,8-naphthalimide for 20-60 min at a wavelength of 200-400 nm and a power of 400-500 W.

[0041] In the preferred technical solution of the present invention, the nano-silicon particles are prepared by ultraviolet irradiation of a precursor obtained by mixing a silicon-oxygen coupling agent and 1,8-naphthalimide for 40 min under the conditions of a wavelength of 365 nm and 400 W.

[0042] In the preferred technical solution of the present invention, the silicon-oxygen coupling agent is 3-aminopropyltrimethoxysilane (APS).

[0043] In the preferred technical solution of the present invention, the microneedles are prepared by a method combining 3D printing and casting with a polydimethylsiloxane (PDMS) negative mold.

[0044] In the preferred technical solution of the present invention, the preparation of the polydimethylsiloxane negative mold includes the following steps: Immerse the 3D printed master template in a polyvinyl alcohol solution with a concentration of 1-5% for 10 h-24 h. After drying, place it upside down and fix it in a petri dish, then add polydimethylsiloxane after defoaming treatment. After curing, peel it off to obtain the polydimethylsiloxane negative mold.

[0045] In the preferred technical solution of the present invention, in the preparation of the polydimethylsiloxane negative mold, the concentration of the polyvinyl alcohol solution is 1-2%.

[0046] In the preferred technical solution of the present invention, the drying temperature in the preparation of the polydimethylsiloxane negative mold is 50°C-100°C, preferably 60°C-80°C.

[0047] In the preferred technical solution of the present invention, the curing temperature in the preparation of the polydimethylsiloxane negative mold is 80°C-100°C, preferably 80°C-90°C.

[0048] In the preferred technical solution of the present invention, the preparation of the drug-loaded needle tip layer includes the following steps. Under stirring (100-200 rpm), in the required amount of nano-complex solution, add polyvinylpyrrolidone according to a mass-volume ratio of polyvinylpyrrolidone:nano-complex solution of 100-200:1 (mg / mL). Inject the mixture solution of polyvinylpyrrolidone and nano-complex solution into the polydimethylsiloxane negative mold. After vacuum degassing, dry for 1-2 h to obtain the drug-loaded needle tip layer.

[0049] In the preferred technical solution of the present invention, in the preparation of the drug-loaded needle tip layer, the mass-volume ratio of polyvinylpyrrolidone:nano-complex solution is 120-150:1 (mg / mL).

[0050] In the preferred technical solution of the present invention, the preparation of the needle body layer includes the following steps. Under the condition of stirring (100 - 200 rpm), mix methacrylated polylysine and methacrylated sodium hyaluronate in a mass ratio of 1:0.25 - 1, then make a 5% solution with PBS (pH 7.4), and then add lithium phenyl - 2,4,6 - trimethylbenzoylphosphinate (LAP) with a concentration of 0.5%. After mixing for 1 - 2 h, filter through a 0.22 μm filter membrane, then inject it into a polydimethylsiloxane negative film, degas under vacuum, and then cure under ultraviolet light and dry for 1 - 2 h to obtain it.

[0051] In the preferred technical solution of the present invention, in the preparation of the needle body layer, the mass ratio of methacrylated polylysine to methacrylated sodium hyaluronate is 1:0.5 - 1.

[0052] In the preferred technical solution of the present invention, the preparation of the drug - loaded reservoir layer includes the following steps: Under the condition of stirring (100 - 200 rpm), in the required amount of nano - complex solution, add polyvinylpyrrolidone to the nano - complex solution in a mass - to - volume ratio of 100 - 200:1 (mg / mL), inject the mixture solution of polyvinylpyrrolidone and nano - complex solution into a polydimethylsiloxane negative mold, degas under vacuum, and then dry for 8 - 10 h to obtain it.

[0053] In the preferred technical solution of the present invention, in the preparation of the drug - loaded reservoir layer, the mass - to - volume ratio of polyvinylpyrrolidone to nano - complex solution is 120 - 150:1 (mg / mL).

[0054] In the preferred technical solution of the present invention, the preparation of the base layer includes the following steps: Under the condition of stirring (100 - 200 rpm), mix polyethylene glycol diacrylate (PEGDA400) and polyethylene glycol diacrylate (PEGDA600) in a mass ratio of 1:1 - 5, add 2,4,6 - trimethylbenzoyl - bis(p - tolyl) phosphine oxide (TMO) with a weight percentage concentration of 0.5%, mix for 0.5 - 1 h, inject the prepared polyethylene glycol diacrylate solution (PEGDA) into a polydimethylsiloxane negative film, and then cure under ultraviolet light to obtain it.

[0055] In the preferred technical solution of the present invention, in the preparation of the base layer, the mass ratio of polyethylene glycol diacrylate (PEGDA400) to polyethylene glycol diacrylate (PEGDA600) is 1:1 - 2.

[0056] In the preferred technical solution of the present invention, in the preparation of the drug - loaded needle tip layer, needle body layer and drug - loaded reservoir layer, the vacuum degassing condition is - 0.1 to - 0.2 MPa for 2 - 10 min, preferably - 0.1 MPa for 2 min.

[0057] In the preferred technical solution of the present invention, in the preparation of either the needle body layer or the base layer, the UV curing conditions are λ = 405 nm, light intensity 25 mW·cm -2 .

[0058] In a preferred technical solution of the present invention, in the preparation of either the drug-loaded needle tip layer or the drug-loaded reservoir layer, the drying temperature is 20°C-30°C, preferably 22-25°C.

[0059] In the preferred technical solution of the present invention, the preparation of the 3D printed microneedles includes the following steps:

[0060] (1) Soak the 3D printing master template in a 1-5% polyvinyl alcohol solution for 10-24 hours. After drying, place it upside down in a culture dish and fix it. Then add defoamed polydimethylsiloxane and cure it at 80-100°C for 2-3 hours. Then peel it off to obtain a polydimethylsiloxane negative mold.

[0061] (2) Under stirring (100-200 rpm), polyvinyl pyrrolidone is added to the required amount of nanocomposite solution at a mass volume ratio of 100-200:1 (mg / mL), and the mixture of polyvinyl pyrrolidone and nanocomposite solution is injected into a polydimethylsiloxane female mold, vacuum degassed, and dried for 1-2 hours to obtain a drug-loaded needle tip layer;

[0062] (3) Under stirring (100-200 rpm), methacrylated polylysine and methacrylated sodium hyaluronate were mixed in a mass ratio of 1:0.25-1, and then prepared into a 5% solution with PBS (pH 7.4), and then added with 0.5% lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP). After mixing for 1-2 hours, the mixture was filtered through a 0.22 μm filter membrane and injected into a polydimethylsiloxane negative membrane. After vacuum degassing, the mixture was UV-cured and dried for 1-2 hours to obtain a needle body layer.

[0063] (4) injecting the mixture solution of polyvinyl pyrrolidone and the nanocomposite into the polydimethylsiloxane anion membrane, vacuum degassing (-0.1 MPa, 2-10 min), and drying for 8-10 h to obtain a drug-loaded reservoir layer;

[0064] (5) Mixing polyethylene glycol diacrylate (PEGDA400) and polyethylene glycol diacrylate (PEGDA600) in a mass ratio of 1:1-5 under stirring (100-200 rpm), adding 0.5% by weight concentration of 2,4,6-trimethylbenzoyl-di(p-tolyl)phosphine oxide, and mixing for 0.5-1 h, injecting the obtained polyethylene glycol diacrylate solution into the polydimethylsiloxane negative film, and UV curing to obtain a base layer;

[0065] (6) Demold to obtain the product.

[0066] Another object of the present invention is to provide a method for preparing 3D printed antibacterial microneedles. The microneedles are biomimetic mushroom-shaped structures made of a drug-loaded tip layer, a needle body layer, a drug-loaded reservoir layer, and a base layer. Among them, the volume ratio of the drug-loaded tip layer: the needle body layer: the drug-loaded reservoir layer: the base layer is 1:1:20 - 50:20 - 50. The method for preparing the microneedles includes the following steps:

[0067] (1) The drug-loaded tip layer is prepared by loading a nano complex with soluble polyvinylpyrrolidone. The mass-volume ratio of soluble polyvinylpyrrolidone to the nano complex solution in the drug-loaded tip layer is 100 - 200:1 (mg / mL);

[0068] (2) The needle body layer is prepared by photocrosslinking methacrylated polylysine and methacrylated sodium hyaluronate. The mass ratio of methacrylated polylysine to methacrylated sodium hyaluronate in the needle body layer is 1:0.25 - 1;

[0069] (3) The drug-loaded reservoir layer is prepared by loading a nano complex with soluble polyvinylpyrrolidone. The mass-volume ratio of soluble polyvinylpyrrolidone to the nano complex solution in the drug-loaded reservoir layer is 100 - 200:1 (mg / mL);

[0070] (4) The base layer is made by photocrosslinking non-degradable polyethylene glycol diacrylate.

[0071] In the preferred technical solution of the present invention, the volume ratio of the drug-loaded tip layer: the needle body layer: the drug-loaded reservoir layer: the base layer is 1:1:30:50.

[0072] In the preferred technical solution of the present invention, the mass-volume ratio of soluble polyvinylpyrrolidone to the nano complex solution in the drug-loaded tip layer is 150:1 (mg / mL).

[0073] In the preferred technical solution of the present invention, the mass ratio of methacrylated polylysine to methacrylated sodium hyaluronate in the needle body layer is 1:0.5 - 1.

[0074] In the preferred technical solution of the present invention, the mass-volume ratio of soluble polyvinylpyrrolidone to the nano complex solution in the drug-loaded reservoir layer is 150:1 (mg / mL).

[0075] In the preferred technical solution of the present invention, the drug-loaded reservoir layer is a circular patch.

[0076] In the preferred technical solution of the present invention, the base layer is a square patch.

[0077] In a preferred technical solution of the present invention, the nano - composite is prepared from an antisense oligonucleotide: dextran - coated nano - silicon particles at a mass ratio of 1:100 - 300. Among them, the antisense oligonucleotide is selected from any one or a combination of nucleotide sequences against Gram - positive bacteria and nucleotide sequences against Gram - negative bacteria. The dextran - coated nano - silicon particles are prepared by covalently binding dextran to the surface of nano - silicon particles, and the dextran is selected from any one or a combination of maltodextrin and diethylaminoethyl dextran.

[0078] In a preferred technical solution of the present invention, the antisense oligonucleotide is composed of a nucleotide sequence against Gram - positive bacteria and a nucleotide sequence against Gram - negative bacteria.

[0079] In a preferred technical solution of the present invention, the nucleotide sequence against Gram - negative bacteria is SEQ ID NO.1.

[0080] In a preferred technical solution of the present invention, the nucleotide sequence against Gram - positive bacteria is SEQ ID NO.2.

[0081] In a preferred technical solution of the present invention, the antisense oligonucleotide is prepared from nucleotide sequence SEQ ID NO.1: nucleotide sequence SEQ ID NO.2 at a mass ratio of 1:2 - 2:1, preferably 1:1.

[0082] In a preferred technical solution of the present invention, the dextran is prepared from maltodextrin: diethylaminoethyl dextran at a mass ratio of 0.5 - 2:1, preferably 1 - 1.5:1.

[0083] In a preferred technical solution of the present invention, the hydrodynamic diameter of the nano - composite is 50 - 300 nm, preferably 80 - 200 nm.

[0084] In a preferred technical solution of the present invention, the diameter of the nano - silicon particles is 2 - 3 nm.

[0085] In a preferred technical solution of the present invention, the hydrodynamic (DLS) diameter of the dextran - coated nano - silicon particles is 5 - 8 nm.

[0086] In a preferred technical solution of the present invention, the encapsulation efficiency of the nano - composite for the antisense oligonucleotide is ≥85%, preferably ≥90%, more preferably ≥95%.

[0087] In a preferred technical solution of the present invention, the uptake rate of any one of the bacteria against Gram - positive bacteria and Gram - negative bacteria for the nano - composite is ≥30%, preferably ≥35%, more preferably ≥40%.

[0088] In a preferred technical solution of the present invention, the polyvinylpyrrolidone is selected from any one or a combination of PVP K30, PVP K60, and PVP K90.

[0089] In the preferred technical solution of the present invention, the concentration of polyvinylpyrrolidone is 10-30%, preferably 13-18%.

[0090] In the preferred technical solution of the present invention, the preparation of the nano-complex includes the following steps: Under the condition of stirring (1000-2000 rpm), the antisense oligonucleotide: dextran nanosilica particles are placed in a mass ratio of 1:100-300 and shaken and mixed at 2°C-8°C for 1-2 h, centrifuged at (5000-10000) g * (15-30) min, and eluted 3-5 times to obtain.

[0091] In the preferred technical solution of the present invention, the preparation temperature of the nano-complex is 4°C-10°C.

[0092] In the preferred technical solution of the present invention, the preparation of the dextran nanosilica particles includes the following steps:

[0093] (1) Mix maltodextrin: diethylaminoethyl dextran in a mass ratio of 0.5-2:1, and add water to make a dextran solution with a concentration of 10-20 mg / ml;

[0094] (2) Under the condition of stirring (100-200 rpm), place the dextran solution and the required amount of nanosilica particle solution at 50-100°C for reaction for 3-6 h, then add the required amount of NaBH4 to the reaction solution and react for 8-12 h, and then ultrafilter and centrifuge the reaction solution 4-6 times to obtain.

[0095] In the preferred technical solution of the present invention, the centrifugation condition is (7500-9000 rpm) * (15-20 min).

[0096] In the preferred technical solution of the present invention, the mass-volume ratio of NaBH4: reaction solution is 1:10-20, preferably 1:15-16.

[0097] In the preferred technical solution of the present invention, the nanosilica particles are prepared by ultraviolet irradiation of a precursor obtained by mixing a siloxane coupling agent and 1,8-naphthalimide for 20-60 min under the conditions of a wavelength of 200-400 nm and 400-500 W.

[0098] In the preferred technical solution of the present invention, the nanosilica particles are prepared by ultraviolet irradiation of a precursor obtained by mixing a siloxane coupling agent and 1,8-naphthalimide for 40 min under the conditions of a wavelength of 365 nm and 400 W.

[0099] In the preferred technical solution of the present invention, the siloxane coupling agent is 3-aminopropyltrimethoxysilane (APS).

[0100] In a preferred technical solution of the present invention, the microneedles are prepared by using a 3D printing combined with a polydimethylsiloxane (PDMS) negative mold replication method.

[0101] In a preferred technical solution of the present invention, the preparation of the polydimethylsiloxane negative mold includes the following steps: Immerse the 3D printing master template in a polyvinyl alcohol solution with a concentration of 1-5% for 10h-24h. After drying, place it upside down and fix it in a petri dish, then add the defoamed polydimethylsiloxane. After curing, peel it off to obtain the polydimethylsiloxane negative mold.

[0102] In a preferred technical solution of the present invention, in the preparation of the polydimethylsiloxane negative mold, the concentration of the polyvinyl alcohol solution is 1-2%.

[0103] In a preferred technical solution of the present invention, in the preparation of the polydimethylsiloxane negative mold, the drying temperature is 50°C-100°C, preferably 60°C-80°C.

[0104] In a preferred technical solution of the present invention, in the preparation of the polydimethylsiloxane negative mold, the curing temperature is 80°C-100°C, preferably 80°C-90°C.

[0105] In a preferred technical solution of the present invention, the preparation of the drug-loaded tip layer includes the following steps. Under stirring conditions (100-200 rpm), in the required amount of the nano-complex solution, add polyvinylpyrrolidone to the nano-complex solution at a mass-to-volume ratio of 100-200:1 (mg / mL). Inject the mixture solution of polyvinylpyrrolidone and the nano-complex solution into the polydimethylsiloxane negative mold. After vacuum degassing, dry for 1-2h to obtain the drug-loaded tip layer.

[0106] In a preferred technical solution of the present invention, in the preparation of the drug-loaded tip layer, the mass-to-volume ratio of polyvinylpyrrolidone to the nano-complex solution is 120-150:1 (mg / mL).

[0107] In a preferred technical solution of the present invention, the preparation of the needle body layer includes the following steps. Under stirring conditions (100-200 rpm), mix methacrylated polylysine and methacrylated sodium hyaluronate at a mass ratio of 1:0.25-1, then make a solution with a concentration of 5% using PBS (pH 7.4), and then add lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) with a concentration of 0.5%. After mixing for 1-2h, filter through a 0.22μm filter membrane, then inject it into the polydimethylsiloxane negative mold. After vacuum degassing, dry for 1-2h and then cure it under ultraviolet light to obtain the needle body layer.

[0108] In the preferred technical solution of the present invention, in the preparation of the needle body layer, the mass ratio of methacrylated polylysine to methacrylated sodium hyaluronate is 1:0.5 - 1.

[0109] In the preferred technical solution of the present invention, the preparation of the drug-loaded reservoir layer includes the following steps: Under the condition of stirring (100 - 200 rpm), in the required amount of the nano-composite solution, polyvinylpyrrolidone is added to the nano-composite solution according to a mass-to-volume ratio of 100 - 200:1 (mg / mL). The mixture solution made of polyvinylpyrrolidone and the nano-composite solution is injected into a polydimethylsiloxane negative mold, degassed under vacuum, and dried for 8 - 10 h to obtain the product.

[0110] In the preferred technical solution of the present invention, in the preparation of the drug-loaded reservoir layer, the mass-to-volume ratio of polyvinylpyrrolidone to the nano-composite solution is 120 - 150:1 (mg / mL).

[0111] In the preferred technical solution of the present invention, the preparation of the base layer includes the following steps: Under the condition of stirring (100 - 200 rpm), poly(ethylene glycol) diacrylate (PEGDA400) and poly(ethylene glycol) diacrylate (PEGDA600) are mixed according to a mass ratio of 1:1 - 5, and 2,4,6-trimethylbenzoyl-diphenylphosphine oxide with a weight percentage concentration of 0.5% is added. After mixing for 0.5 - 1 h, the prepared poly(ethylene glycol) diacrylate solution is injected into a polydimethylsiloxane negative mold. After ultraviolet curing, the product is obtained.

[0112] In the preferred technical solution of the present invention, in the preparation of the base layer, the mass ratio of poly(ethylene glycol) diacrylate (PEGDA400) to poly(ethylene glycol) diacrylate (PEGDA600) is 1:1 - 2.

[0113] In the preferred technical solution of the present invention, in the preparation of the drug-loaded needle tip layer, the needle body layer and the drug-loaded reservoir layer, the vacuum degassing condition is -0.1 to -0.2 MPa for 2 - 10 min, preferably -0.1 MPa for 2 min.

[0114] In the preferred technical solution of the present invention, in any preparation of the needle body layer and the base layer, the ultraviolet curing condition is λ = 405 nm and the light intensity is 25 mW·cm -2 .

[0115] In the preferred technical solution of the present invention, in any preparation of the drug-loaded needle tip layer and the drug-loaded reservoir layer, the drying temperature is 20°C - 30°C, preferably 22 - 25°C.

[0116] In the preferred technical solution of the present invention, the preparation of the 3D printed microneedles includes the following steps:

[0117] (1) Immerse the 3D printing master template in a polyvinyl alcohol solution with a concentration of 1-5% for 10h-24h. After drying, place it upside down and fix it in a petri dish. Then add polydimethylsiloxane after defoaming treatment and cure it at 80-100°C for 2-3h, and then peel it off to obtain a polydimethylsiloxane negative mold;

[0118] (2) Under the condition of stirring (100-200rpm), in the required amount of nano-composite solution, add polyvinylpyrrolidone to the nano-composite solution at a mass-volume ratio of 100-200:1 (mg / mL). Inject the mixture solution made of polyvinylpyrrolidone and nano-composite solution into the polydimethylsiloxane negative mold. After vacuum degassing, dry it for 1-2h to obtain a drug-loaded needle tip layer;

[0119] (3) Under the condition of stirring (100-200rpm), mix methacrylated polylysine and methacrylated sodium hyaluronate at a mass ratio of 1:0.25-1, and then make a solution with a concentration of 5% using PBS (pH 7.4). Then add lithium phenyl-2,4,6-trimethylbenzoylphosphate (LAP) with a concentration of 0.5%, mix for 1-2h, filter through a 0.22μm filter membrane, and then inject it into the polydimethylsiloxane negative membrane. After vacuum degassing, cure it under ultraviolet light and dry it for 1-2h to obtain a needle body layer;

[0120] (4) Inject the mixture solution made of polyvinylpyrrolidone and nano-composite solution into the polydimethylsiloxane negative membrane. After vacuum degassing (-0.1MPa, 2-10min), dry it for 8-10h to obtain a drug-loaded reservoir layer;

[0121] (5) Under the condition of stirring (100-200rpm), mix polyethylene glycol diacrylate (PEGDA400) and polyethylene glycol diacrylate (PEGDA600) at a mass ratio of 1:1-5, add 2,4,6-trimethylbenzoyl-bis(p-tolyl)phosphine oxide with a weight percentage concentration of 0.5%, mix for 0.5-1h, and inject the obtained polyethylene glycol diacrylate solution into the polydimethylsiloxane negative membrane. After curing under ultraviolet light, obtain a base layer;

[0122] (6) Demold to obtain the product.

[0123] Another object of the present invention is to provide a nano - composite, which is prepared from antisense oligonucleotides (ASOs) and dextran - coated silicon nanoparticles (GP - SiNPs) at a mass ratio of 1:100 - 300. Among them, the antisense oligonucleotides are selected from any one or a combination of nucleotide sequences against Gram - positive bacteria and nucleotide sequences against Gram - negative bacteria. The dextran - coated silicon nanoparticles are prepared by covalently binding dextran (GP) to the surface of silicon nanoparticles (SiNPs). The dextran is selected from any one or a combination of maltodextrin (MD) and diethyl - aminoethyl - dextran (DEAE).

[0124] In a preferred technical solution of the present invention, the antisense oligonucleotides are composed of a nucleotide sequence against Gram - positive bacteria and a nucleotide sequence against Gram - negative bacteria.

[0125] In a preferred technical solution of the present invention, the nucleotide sequence against Gram - negative bacteria is SEQ ID NO.1.

[0126] In a preferred technical solution of the present invention, the nucleotide sequence against Gram - positive bacteria is SEQ ID NO.2.

[0127] In a preferred technical solution of the present invention, the antisense oligonucleotides are prepared from SEQ ID NO.1:SEQ ID NO.2 at a mass ratio of 1:2 - 2:1, preferably 1:1.

[0128] In a preferred technical solution of the present invention, the dextran is prepared from maltodextrin and diethyl - aminoethyl - dextran at a mass ratio of 0.5 - 2:1, preferably 1 - 1.5:1.

[0129] In a preferred technical solution of the present invention, the hydrated particle size of the nano - composite is 50 - 300 nm, preferably 80 - 200 nm.

[0130] In a preferred technical solution of the present invention, the diameter of the silicon nanoparticles is 2 - 3 nm.

[0131] In a preferred technical solution of the present invention, the hydrodynamic diameter (Dynamic Light Scattering, DLS) of the dextran - coated silicon nanoparticles is 5 - 8 nm.

[0132] In a preferred technical solution of the present invention, the encapsulation efficiency of the nano - composite for antisense oligonucleotides is ≥85%, preferably ≥90%, more preferably ≥95%.

[0133] In a preferred technical solution of the present invention, the uptake rate of any one of the anti - Gram - positive bacteria and anti - Gram - negative bacteria for the nano - composite is ≥30%, preferably ≥35%, more preferably ≥40%.

[0134] In a preferred technical solution of the present invention, the polyvinylpyrrolidone is selected from any one or a combination of PVP K30, PVP K60, and PVP K90.

[0135] In a preferred technical solution of the present invention, the concentration of the polyvinylpyrrolidone is 10-30%, preferably 13-18%.

[0136] In a preferred technical solution of the present invention, the preparation of the nano-complex includes the following steps: Under stirring (1000-2000 rpm), the antisense oligonucleotide: dextran nanosilica particles are placed in a mass ratio of 1:100-300 at 2°C-8°C and shaken and mixed for 1-2 h, centrifuged at (5000-10000) g for (15-30) min, and eluted 3-5 times to obtain the nano-complex.

[0137] In a preferred technical solution of the present invention, the preparation temperature of the nano-complex is 4°C-10°C.

[0138] In a preferred technical solution of the present invention, the preparation of the dextran nanosilica particles includes the following steps:

[0139] (1) Mix maltodextrin: diethylaminoethyl dextran in a mass ratio of 0.5-2:1, and add water to make a dextran solution with a concentration of 10-20 mg / ml;

[0140] (2) Under stirring (100-200 rpm), place the dextran solution and the required amount of nanosilica particle solution at 50-100°C for reaction for 3-6 h, then add the required amount of NaBH4 to the reaction solution and react for 8-12 h, and then ultrafilter and centrifuge the reaction solution 4-6 times to obtain the dextran nanosilica particles.

[0141] In a preferred technical solution of the present invention, the centrifugation conditions are (7500-9000 rpm)×(15-20 min).

[0142] In a preferred technical solution of the present invention, the mass-volume ratio of NaBH4 to the reaction solution is 1:10-20, preferably 1:15-16.

[0143] In a preferred technical solution of the present invention, the nanosilica particles are prepared by ultraviolet irradiation of a precursor obtained by mixing a siloxane coupling agent and 1,8-naphthalimide for 20-60 min at a wavelength of 200-400 nm and 400-500 W.

[0144] In a preferred technical solution of the present invention, the nanosilica particles are prepared by ultraviolet irradiation of a precursor obtained by mixing a siloxane coupling agent and 1,8-naphthalimide for 40 min at a wavelength of 365 nm and 400 W.

[0145] In a preferred technical solution of the present invention, the siloxane coupling agent is 3-aminopropyltrimethoxysilane (APS).

[0146] Another object of the present invention is to provide a method for preparing a nano-complex, which is made of antisense oligonucleotides (ASOs): dextran nanosilicon particles (GP-SiNPs) in a mass ratio of 1:100 - 300. Among them, the antisense oligonucleotides are selected from any one or a combination of nucleotide sequences against Gram-positive bacteria and nucleotide sequences against Gram-negative bacteria. The dextran nanosilicon particles are made by covalently binding dextran (GP) to the surface of nanosilicon particles (SiNPs). The dextran is selected from any one or a combination of maltodextrin (MD) and diethylaminoethyl dextran (DEAE). The preparation of the nano-complex includes the following steps: Under the condition of stirring (1000 - 2000 rpm), place antisense oligonucleotides: dextran nanosilicon particles in a mass ratio of 1:100 - 300 at 2°C - 8°C and shake and mix for 1 - 2 h, centrifuge at (5000 - 10000) g for (15 - 30) min, and elute 3 - 5 times to obtain the product.

[0147] In a preferred technical solution of the present invention, the preparation temperature of the nano-complex is 4°C - 10°C.

[0148] In a preferred technical solution of the present invention, the preparation of the dextran nanosilicon particles includes the following steps:

[0149] (1) Mix maltodextrin: diethylaminoethyl dextran in a mass ratio of 0.5 - 2:1, and add water to make a dextran solution with a concentration of 10 - 20 mg / ml.

[0150] (2) Under the condition of stirring (100 - 200 rpm), place the dextran solution and the required amount of nanosilicon particle solution at 50 - 100°C and react for 3 - 6 h, then add the required amount of NaBH4 to the reaction solution and react for 8 - 12 h, and then ultrafilter and centrifuge the reaction solution 4 - 6 times to obtain the product.

[0151] In a preferred technical solution of the present invention, the centrifugation condition is (7500 - 9000 rpm) * (15 - 20 min).

[0152] In a preferred technical solution of the present invention, the mass-volume ratio of NaBH4 to the reaction solution is 1:10 - 20, preferably 1:15 - 16.

[0153] In a preferred technical solution of the present invention, the nano-silicon particles are prepared by subjecting a precursor obtained by mixing a silicon-oxygen coupling agent and 1,8-naphthalimide to ultraviolet irradiation for 20 - 60 min under the conditions of a wavelength of 200 - 400 nm and 400 - 500 W.

[0154] In a preferred technical solution of the present invention, the nano-silicon particles are prepared by subjecting a precursor obtained by mixing a silicon-oxygen coupling agent and 1,8-naphthalimide to ultraviolet irradiation for 40 min under the conditions of a wavelength of 365 nm and 400 W.

[0155] In a preferred technical solution of the present invention, the silicon-oxygen coupling agent is 3-aminopropyltrimethoxysilane (APS).

[0156] In a preferred technical solution of the present invention, the antisense oligonucleotide is composed of a nucleotide sequence against Gram-positive bacteria and a nucleotide sequence against Gram-negative bacteria.

[0157] In a preferred technical solution of the present invention, the nucleotide sequence against Gram-negative bacteria is SEQ ID NO.1.

[0158] In a preferred technical solution of the present invention, the nucleotide sequence against Gram-positive bacteria is SEQ ID NO.2.

[0159] In a preferred technical solution of the present invention, the antisense oligonucleotide is prepared from SEQ ID NO.1:SEQ ID NO.2 in a mass ratio of 1:2 - 2:1, preferably 1:1.

[0160] In a preferred technical solution of the present invention, the dextran is prepared from maltodextrin:diethylaminoethyl dextran in a mass ratio of 0.5 - 2:1, preferably 1 - 1.5:1.

[0161] In a preferred technical solution of the present invention, the hydrodynamic diameter of the nano-complex is 50 - 300 nm, preferably 80 - 200 nm.

[0162] In a preferred technical solution of the present invention, the diameter of the nano-silicon particles is 2 - 3 nm.

[0163] In a preferred technical solution of the present invention, the hydrodynamic (DLS) diameter of the dextran nano-silicon particles is 5 - 8 nm.

[0164] In a preferred technical solution of the present invention, the encapsulation efficiency of the nano-complex for the antisense oligonucleotide is ≥85%, preferably ≥90%, more preferably ≥95%.

[0165] In a preferred technical solution of the present invention, the uptake rate of any one of the anti-Gram-positive bacteria and anti-Gram-negative bacteria for the nano-complex is ≥30%, preferably ≥35%, more preferably ≥40%.

[0166] In a preferred technical solution of the present invention, the polyvinylpyrrolidone is selected from any one or a combination of PVP K30, PVP K60, and PVP K90.

[0167] In a preferred technical solution of the present invention, the concentration of the polyvinylpyrrolidone is 10-30%, preferably 13-18%.

[0168] Another object of the present invention is to provide the application of the nanocomposite or its 3D printed antibacterial microneedles described in the present invention in antibacterial treatment.

[0169] In a preferred technical solution of the present invention, the bacteria are selected from any one or a combination of Gram-positive bacteria and Gram-negative bacteria.

[0170] In a preferred technical solution of the present invention, the bacteria are selected from any one or a combination of Staphylococcus aureus, Staphylococcus haemolyticus, Escherichia coli, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Proteus, Klebsiella pneumoniae, and Enterobacter cloacae.

[0171] In a preferred technical solution of the present invention, the nanocomposite acts on any one or a combination of the acpP gene of Escherichia coli, the ftsZ gene of Staphylococcus aureus, the ftsZ gene of filamentous temperature-sensitive protein Z, the acpP gene of acyl carrier protein, the mecA gene of methicillin-resistant gene, and the YycFG gene of two-component regulatory gene.

[0172] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between a liquid and a solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between a solid and a liquid, the percentage is weight / volume percentage; and the rest is weight / weight percentage.

[0173] Unless otherwise specified, the present invention uses a Malvern nanoparticle size analyzer to detect particle size, polydispersity index PDI, and Zeta potential.

[0174] Unless otherwise specified, the present invention uses the following method to detect the encapsulation rate of the nanocomposite for the antisense oligonucleotide: Place the nanocomposite sample to be tested in an ultrafiltration tube (MW cut-off 10 kDa; Millipore) and centrifuge (8000 rpm * 30 min, 4 °C) to remove the completely bound antisense oligonucleotide. Take the supernatant and use a Nanodrop ultra-micro spectrophotometer to measure the mass of the free antisense oligonucleotide. Detect in parallel three times and calculate the encapsulation rate (Embedding ratio, ER).

[0175] ER = (W0 - W) / W0 × 100%

[0176] Among them, W0 is the initial mass of the antisense oligonucleotide, and W is the mass of the antisense oligonucleotide in the supernatant.

[0177] Compared with the prior art, the present invention has the following beneficial effects:

[0178] 1. Based on the "Trojan horse" strategy, the present invention innovatively designs a biomimetic nano-complex with a pseudo-glycogen shell to simulate the natural glycogen metabolism pathway of bacteria. First, maltodextrin is used as a specific ligand for the bacterial ABC transporter to induce the active uptake of nanoparticles by bacteria. Second, the protonated tertiary amine group of diethylaminoethyl dextran is used, which is positively charged at physiological pH and has an electrostatic interaction with the negatively charged antisense oligonucleotide to achieve efficient nucleic acid loading. Third, 3-aminopropyltrimethoxysilane (APS) silicon precursor and 1,8-naphthalimide undergo a photocatalytic reaction under ultraviolet irradiation to prepare nano-silicon particles (SiNPs) with characteristics such as photo-stability, pH stability (pH 4-10), ultra-small nano-scale, and surface modification with amino groups as the core of the dextran nano-silicon particles. Fourth, maltodextrin and diethylaminoethyl dextran are covalently bound to the amino groups on the surface of the nano-silicon particles, and then the chemical bond is fixed by reduction with sodium borohydride (NaBH4) to construct dextran nano-silicon particles with high drug loading, specific uptake ability, and stability. Fifth, the specific recognition of the bacterial ABC transporter for sugar ligands is utilized, and the silicon-sugar ratio and the ratio of maltodextrin / diethylaminoethyl dextran are regulated to optimize the drug loading performance and stability of the nanoparticles. Sixth, the antisense oligonucleotide is electrostatically adsorbed on the surface of the dextran nano-silicon particles, thereby achieving specific recognition and efficient uptake of Gram-negative bacteria (such as Escherichia coli, etc.) and Gram-positive bacteria (such as Staphylococcus aureus, etc.). For the first time, a new type of "Trojan horse" nano-complex with high efficient targeting delivery efficiency for both Gram-positive and Gram-negative bacteria, stable loading of antisense oligonucleotides, and excellent antibacterial efficacy is developed, breaking through the problem of antisense oligonucleotide antibacterial therapy.

[0179] 2. The present invention adopts a 3D printing polydimethylsiloxane molding method to integrally prepare bionic mushroom-shaped microneedles made of a drug-loaded tip layer, a drug-loaded needle body layer, a drug-loaded reservoir layer, and a base layer. The microneedles have excellent mechanical properties (microneedle fracture rate ≤ 10%) and puncture performance. Among them, the drug-loaded tip layer has excellent puncture ability and adhesiveness, can quickly and effectively open the biological membrane and bacterial barrier, and realize the rapid release of antibacterial drugs; the needle body layer swells after absorbing tissue fluid, continuously opens the drug delivery channel, and promotes the targeted drug delivery and long-term release of active ingredients; the drug-loaded reservoir layer long-term releases antibacterial drugs; the base layer effectively absorbs tissue exudate, protects the wound and promotes wound healing, and can be completely peeled off to ensure that there is no polymer residue on the wound surface. The array structure of the microneedles accurately punctures the wound surface and the stratum corneum of the skin, creating hundreds of micron-sized drug delivery channels in and around the wound and wound-infected tissues, significantly increasing the contact area between the drug and the hidden infection site, and targeting the antisense oligonucleotide drug to the deep infection focus. Moreover, the microneedle drug delivery system of the present invention is conducive to achieving self-administration at the wound site, stimulating the generation of collagen and elastin and angiogenesis, inducing the release of growth factors to promote wound healing, realizing the precise release, efficient uptake of drugs, and the cascade effect of "tissue penetration - bacterial targeting - gene silencing", significantly shortening the wound healing time, significantly improving the targeted delivery efficiency, bioavailability, safety and effectiveness of antibacterial drugs, and overcoming the problem that nucleic acid drugs are difficult to be used for anti-wound infection.

[0180] 3. The present invention scientifically screens the preparation processes and parameters of the nano-complex and the microneedles, and combines the PDMS molding method to prepare the microneedles, significantly improving the accuracy, durability and mechanical strength of the PDMS negative mold (tip fracture rate < 10%), and effectively solving the problems of large demolding stress and high tip residue rate existing in the PDMS replication method.

[0181] 4. The preparation method of the present invention has the advantages of simple operation, high yield, better cost, wide applicability to the population, and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0182] Figure 1 TEM analysis results of the nano-complex of the present invention;

[0183] Figure 2 Particle size stability study of the nano-complex of the present invention;

[0184] Figure 3 Biological safety investigation of the nano-complex of the present invention, a. Influence of the nano-complex on the growth activity of L929 cells; b. Research results on the hemolytic effect of the nano-complex; c. Detection results on the blood cell compatibility of the nano-complex;

[0185] Figure 4Study on the antibacterial activity of the nano - composite of the present invention, a. Escherichia coli; b. Staphylococcus aureus;

[0186] Figure 5 Study on the uptake efficiency of the nano - composite of the present invention in bacteria, a. Escherichia coli, b. Staphylococcus aureus;

[0187] Figure 6 Investigation on the bacterial mRNA degradation mediated by the nano - composite of the present invention, a. Analyze the content of mEc acpP by semi - quantitative gel electrophoresis and real - time fluorescence quantitative PCR; b. Analyze the content of mSau fmhB by semi - quantitative gel electrophoresis and real - time fluorescence quantitative PCR;

[0188] Figure 7 Performance characterization of the 3D - printed microneedles of the present invention;

[0189] Figure 8 Investigation on the mechanical properties of the 3D - printed microneedles of the present invention;

[0190] Figure 9 Investigation on the in vitro drug release of the 3D - printed microneedles of the present invention;

[0191] Figure 10 Study on the in - vivo antibacterial and wound - healing promotion properties of the 3D - printed microneedles of the present invention. Detailed implementation manners

[0192] The present invention will be specifically described below in conjunction with the embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and do not limit the essence of the present invention.

[0193] The designed nucleic acid sequences (see Table 1) in the detailed implementation manners were entrusted to Beijing Tsingke Biotechnology Co., Ltd. to be prepared by solid - phase synthesis method, purified by high - performance liquid chromatography, and the molecular weight was confirmed by matrix - assisted laser desorption / ionization time - of - flight mass spectrometer.

[0194] Table 1 Nucleic acid sequences

[0195]

[0196] The preparation of the polydimethylsiloxane negative mold in the detailed implementation manners includes the following steps:

[0197] 1) Preparation of the 3D - printed microneedle master template

[0198] Install the slicing software BMF 3D slicer V1.6.4 on the industrial control computer of the printer, import the.stl file of the microneedle three-dimensional model, slice the three-dimensional model, set the slice layer thickness to 10 μm, and the XY plane accuracy to 10 μm to obtain.png format pictures. The program will automatically name the sliced two-dimensional pictures in sequence. In the 3D Printing System S-140S1.0.1 software of the printer, select the stitching printing mode, import the.png format pictures, and set the printing parameters: exposure time (Exposure time, Exp-t), exposure intensity (Intensity Intst), number of scraper movements (Scra), etc. After saving the printing parameters, select the HLT resin of Shenzhen Magic Square Precision to prepare the 3D printing master template.

[0199] After soaking the prepared 3D printing master template in ethanol for 1 h, perform ultraviolet secondary curing for 2 - 5 min, and then soak it in ethanol for 24 h to obtain the 3D printing microneedle master template:

[0200] Tip layer: needle length 1100 μm, bottom diameter 450 μm, tip spacing 800 μm, number of arrays 113, distribution area 10.5 mm * 10.5 mm;

[0201] Base layer: first order: 11 * 11 mm (circular), second order: 13 * 13 mm (square), groove depth of both the first order and the second order is 1 mm; [[ID=I0]]

[0202] 2) Preparation of the polydimethylsiloxane negative mold: Immerse the 3D printing master template in a 1% polyvinyl alcohol solution for 12 h, dry it at 60 °C, then invert it and fix it in a 35 mm petri dish. After defoaming the polydimethylsiloxane, add it to the petri dish and cure it at 80 °C for 2 h, then peel it off to obtain it.

[0203] Preparation of the mixture solution of polyvinylpyrrolidone and the nanocomplex in the specific embodiment: Take 30 mL of the nanocomplex solution in Example 5, and under stirring (180 rpm), add 4.5 g of polyvinylpyrrolidone (PVP K90) and mix for 2 h to obtain it.

[0204] Preparation of the methacrylated polylysine / methacrylated sodium hyaluronate solution in the specific embodiment: Under stirring (180 rpm), dissolve 5% methacrylated polylysine and 5% methacrylated sodium hyaluronate in PBS (pH 7.4), add 0.5% lithium phenyl - 2,4,6 - trimethylbenzoylphosphonate (LAP) photoinitiator, mix for 2 h, and filter the reaction solution through a 0.22 μm filter membrane to obtain 9 mL of methacrylated polylysine / methacrylated sodium hyaluronate solution, which is stored in the dark at 4 °C for standby.

[0205] Preparation of polyethylene glycol diacrylate (PEGDA) solution in the specific implementation manner: Under the condition of stirring (180 rpm), mix polyethylene glycol diacrylate (PEGDA400): polyethylene glycol diacrylate (PEGDA600) according to a mass ratio of 1:1, add 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TMO) with a weight percentage concentration of 0.5%, react for 30 min, and 30 mL of polyethylene glycol diacrylate (PEGDA) solution is obtained, which is stored in the dark for standby.

[0206] Example 1 Preparation of the nano-silicon particles of the present invention

[0207] The preparation of the nano-silicon particles includes the following steps:

[0208] 1) Dissolve 10.0 g of sodium citrate and 5.0 g of 1,8-naphthalenedicarboximide in 900 mL of water. After stirring and mixing evenly (200 rpm * 10 min), add 100 mL of 3-aminopropyltrimethoxysilane (APS), and continuously stir at 200 rpm for 7 days to obtain a precursor solution;

[0209] 2) Continuously irradiate the obtained precursor solution with ultraviolet light at a wavelength of 365 nm and a condition of 400 - 500 W for 40 min. After natural cooling, filter the obtained nano-silicon particle solution through a 0.45 μm polytetrafluoroethylene filter membrane, and then dialyze the collected supernatant with Milli-Q water (MWCO, 1000, Spectra / Pro). Then, concentrate the obtained purified nano-silicon particle solution to 100 mL under the conditions of 50 °C * 150 rpm by rotary evaporation, and store the obtained refined mother liquor of the nano-silicon particles in the dark at 4 °C.

[0210] After detection, the hydrodynamic diameter of the nano-silicon particles is 2.033 ± 0.5683 nm, and the Zeta potential is +2.44 ± 0.31 mV.

[0211] Example 2 Preparation of the dextran nano-silicon particles of the present invention

[0212] The preparation of the dextran nano-silicon particles includes the following steps:

[0213] 1) Uniformly mix maltodextrin and diethylaminoethyl dextran according to a mass ratio of 2:1, add water, and obtain a dextran solution with a concentration of 20 mg / mL;

[0214] 2) Under stirring (100 rpm), mix the dextran solution at 20 mg / mL and the nano-silicon particle solution at a concentration of 20 mg / mL in equal volumes, then place it under shaking at 60 °C for 5 h to obtain a reaction solution. Then, add NaBH4 according to the mass-volume ratio of NaBH4:reaction solution being 1:16 (mg / mL) and react for 12 h. Place the prepared dextran nano-silicon particle solution in a 3K ultrafiltration tube and ultrafilter it 6 times (7500 rpm * 15 min) to obtain the purified dextran nano-silicon particle solution.

[0215] Detected by a dynamic light scattering instrument and a transmission electron microscope (TEM), the dextran nano-silicon particles are spherical with a uniform particle size distribution, having a hydrated particle size of 7.85 ± 1.90 nm and a Zeta potential of +2.78 ± 0.5 mV.

[0216] Example 3 Preparation of the dextran nano-silicon particles of the present invention

[0217] The preparation of the dextran nano-silicon particles includes the following steps:

[0218] 1) Uniformly mix maltodextrin and diethylaminoethyl dextran in a mass ratio of 1:1, then add water to prepare a dextran solution at 20 mg / mL;

[0219] 2) Under stirring (100 rpm), mix the dextran solution at 20 mg / mL and the nano-silicon particle solution at a concentration of 20 mg / mL in equal volumes, then place it under shaking at 60 °C for 5 h to obtain a reaction solution. Then, add NaBH4 according to the mass-volume ratio of NaBH4:reaction solution being 1:16 (mg / mL) and react for 12 h. Place the prepared dextran nano-silicon particle solution in a 3K ultrafiltration tube and ultrafilter it 6 times (75,00 rpm * 15 min) to obtain the purified dextran nano-silicon particle solution.

[0220] Detected by a dynamic light scattering instrument, the dextran nano-silicon particles are spherical with a uniform particle size distribution, having a hydrated particle size of 5.01 ± 1.56 nm and a Zeta potential of +4.34 ± 1.15 mV.

[0221] Example 4 Preparation of the dextran nano-silicon particles of the present invention

[0222] The preparation of the dextran nano-silicon particles includes the following steps:

[0223] 1) Uniformly mix maltodextrin and diethylaminoethyl dextran in a mass ratio of 1:2, then add water to prepare a dextran solution at 20 mg / mL;

[0224] (2) Under stirring (100 rpm), mix an equal volume of a 20 mg / mL dextran solution and a 20 mg / mL nano-silicon particle solution, place the mixture under shaking at 60 °C for 5 h to obtain a reaction solution, then add NaBH4 according to the mass-to-volume ratio of NaBH4:reaction solution of 1:16 (mg / mL), react for 12 h, place the prepared dextran nano-silicon particle solution in a 3K ultrafiltration tube (7500 rpm * 15 min) and ultrafilter 6 times to obtain a purified dextran nano-silicon particle solution.

[0225] Detected by a dynamic light scattering instrument, the dextran nano-silicon particles are spherical with a uniform particle size distribution, having a hydrodynamic diameter of 6.75 ± 1.41 nm and a Zeta potential of +4.71 ± 0.58 mV.

[0226] Example 5 Preparation of the nano-composite of the present invention

[0227] The preparation of the nano-composite includes the following steps:

[0228] (1) Dissolve nucleic acid sequence NO.1 (Ec acpP) and nucleic acid sequence NO.2 (Sau ftsZ) in diethyl pyrocarbonate (DEPC) water according to a mass ratio of 1:1, measure its absorbance at 260 nm using a Nanodrop ultra-micro spectrophotometer to obtain an antisense oligonucleotide solution with an OD of 0.4, then add the dextran nano-silicon particle solution of Example 2 according to the mass ratio of antisense oligonucleotide to dextran nano-silicon particle of 1:200, mix evenly, and incubate at 4 °C and 1000 rpm for 12 h to obtain a crude product solution;

[0229] (2) Centrifuge the crude product solution through a Nanosep centrifugation device (M W Retain, 10 kDa, Millipore) by centrifugation (5000×g * 15 min), and elute 5 times with PBS to obtain 100 μL of a nano-composite solution.

[0230] Detected, the encapsulation efficiency of the nano-composite is 96.68%, the hydrodynamic diameter is 113.87 nm, the PDI is 0.5507, and the Zeta potential is -2.55 ± 0.05 mV.

[0231] Example 6 Synthesis of the nano-composite of the present invention

[0232] The preparation of the nano-composite includes the following steps:

[0233] (1) Dissolve nucleic acid sequence NO.1 (Ec acpP) and nucleic acid sequence NO.2 (Sau ftsZ) in diethyl pyrocarbonate (DEPC) water at a mass ratio of 1:1. Use a Nanodrop ultra-micro spectrophotometer to measure the absorbance at 260 nm to prepare an antisense oligonucleotide solution with an OD of 0.4. Add the dextran nanosilica particle solution of Example 3 according to the mass ratio of antisense oligonucleotide to dextran nanosilica particles of 1:200. After mixing evenly, incubate at 4 °C and 1000 rpm for 12 h to obtain a crude product solution;

[0234] (2) Centrifuge the crude product solution through a Nanosep centrifugal device (M W Retention, 10 kDa, Millipore) by centrifugation (5000×g * 15 min), and elute 5 times with PBS to prepare 100 μL of a nanocomplex solution.

[0235] After detection, the encapsulation efficiency of the nanocomplex is 97.49%, the hydrated particle size is 82.11 nm, the PDI is 0.3777, and the Zeta potential is -3.67 ± 0.26 mV.

[0236] Example 7 Synthesis of the nanocomplex of the present invention

[0237] The preparation of the nanocomplex includes the following steps:

[0238] (1) Dissolve nucleic acid sequence NO.1 (Ec acpP) and nucleic acid sequence NO.2 (Sau ftsZ) in diethyl pyrocarbonate (DEPC) water at a mass ratio of 1:1. Use a Nanodrop ultra-micro spectrophotometer to measure the absorbance at 260 nm to prepare an antisense oligonucleotide solution with an OD of 0.4. According to the mass ratio of antisense oligonucleotide to dextran nanosilica particles of 1:200, add the dextran nanosilica particle solution of Example 4. After mixing evenly, incubate at 4 °C and 1000 rpm for 12 h to obtain a crude product solution;

[0239] (2) Centrifuge the crude product solution through a Nanosep centrifugal device (M W Retention, 10 kDa, Millipore) by centrifugation (5000×g * 15 min), and elute 5 times with PBS to prepare 100 μL of a nanocomplex solution.

[0240] After detection, the encapsulation efficiency of the nanocomplex is 97.44%, the hydrated particle size is 84.22 nm, the PDI is 0.304, and the Zeta potential is -4.55 ± 0.28 mV.

[0241] Example 8 Synthesis of the nanocomplex of the present invention

[0242] The preparation of the nano - composite includes the following steps:

[0243] (1) Nucleic acid sequence NO.1 (Ec acpP) and nucleic acid sequence NO.2 (Sau ftsZ) are dissolved in diethyl pyrocarbonate (DEPC) water at a mass ratio of 1:1. The absorbance at 260 nm is measured using a Nanodrop ultra - micro spectrophotometer to prepare an antisense oligonucleotide solution with an OD of 0.4. According to the mass ratio of antisense oligonucleotide to dextran - nanosilica particles of 1:100, the dextran - nanosilica particle solution of Example 2 is added. After mixing evenly, it is incubated at 4 °C and 1000 rpm for 12 h to obtain a crude product solution;

[0244] (2) The crude product solution is centrifuged (5000×g * 15 min) through a Nanosep centrifugation device (M W cutoff, 10 kDa, Millipore) and eluted 5 times with PBS to prepare 100 μL of nano - composite solution.

[0245] Example 9 Synthesis of the nano - composite of the present invention

[0246] The preparation of the nano - composite includes the following steps:

[0247] (1) Nucleic acid sequence NO.1 (Ec acpP) and nucleic acid sequence NO.2 (Sau ftsZ) are dissolved in diethyl pyrocarbonate (DEPC) water at a mass ratio of 1:1. The absorbance at 260 nm is measured using a Nanodrop ultra - micro spectrophotometer to prepare an antisense oligonucleotide solution with an OD of 0.4. According to the mass ratio of antisense oligonucleotide to dextran - nanosilica particles of 1:300, the dextran - nanosilica particle solution of Example 2 is added. After mixing evenly, it is incubated at 4 °C and 1000 rpm for 12 h to obtain a crude product solution;

[0248] (2) The crude product solution is centrifuged (5000×g * 15 min) through a Nanosep centrifugation device (M W cutoff, 10 kDa, Millipore) and eluted 5 times with PBS to prepare 100 μL of nano - composite solution.

[0249] Example 10 Synthesis of the nano - composite of the present invention

[0250] The preparation of the nano - composite includes the following steps:

[0251] (1) Dissolve nucleic acid sequence NO.5 (Ec acpP-cy5) and nucleic acid sequence NO.6 (Sau ftsZ-cy5) in diethyl pyrocarbonate (DEPC) water at a mass ratio of 1:1. Use a Nanodrop ultra-micro spectrophotometer to measure the absorbance at 260 nm to prepare an antisense oligonucleotide solution with an OD of 0.4. According to the mass ratio of antisense oligonucleotide to dextran nanosilica particles of 1:200, add the dextran nanosilica particle solution of Example 2, mix evenly, and incubate at 4 °C and 1000 rpm for 12 h to obtain a crude product solution;

[0252] (2) The crude product solution is centrifuged (5000×g * 15 min) through a Nanosep centrifugal device (M W retention, 10 kDa, Millipore), and eluted 5 times with PBS to prepare 100 μL of a nanocomplex solution.

[0253] Example 11 Preparation of 3D printed microneedles of the present invention

[0254] The preparation of 3D printed microneedles includes the following steps:

[0255] (1) Inject the mixture solution of polyvinylpyrrolidone and nanocomplex into a polydimethylsiloxane negative mold. After vacuum degassing treatment (-0.1 MPa, 2 min), scrape off the remaining solution and bubbles, and dry at room temperature for 2 h to obtain a drug-loaded tip layer with a volume of 10 μL;

[0256] (2) Inject the methacrylated polylysine / methacrylated sodium hyaluronate solution into the polydimethylsiloxane negative mold. After vacuum degassing treatment (-0.1 MPa, 2 min), dry at room temperature for 2 h, and then perform ultraviolet curing (λ = 405 nm, light intensity 25 mW·cm -2 , 5 cm away from the mold surface) for 90 s, and then cool to room temperature to obtain a needle body layer with a volume of 10 μL;

[0257] (3) Inject the mixture solution of polyvinylpyrrolidone and nanocomplex into the polydimethylsiloxane negative mold. After vacuum degassing treatment (-0.1 MPa, 2 min), scrape off the remaining solution and bubbles, and dry at room temperature for 8 h to obtain a drug-loaded reservoir layer with a drug-loaded volume of 200 μL;

[0258] (4) Inject the polyethylene glycol diacrylate solution into the polydimethylsiloxane negative mold, and perform ultraviolet curing (λ = 405 nm, light intensity 25 mW·cm -2 , 5 cm away from the mold surface) for 90 s, and then cool to room temperature to obtain a base layer with a volume of 500 μL;

[0259] (5) After standing for 1 - 2 h, demold to obtain the product.

[0260] Comparative Example 1 Synthesis of nanocomposite

[0261] The preparation of the nanocomposite comprises the following steps:

[0262] (1) Under stirring (1000 rpm), nucleic acid sequence NO.3 (Ec acpP’) and nucleic acid sequence NO.4 (SauftsZ’) are dissolved in diethyl pyrocarbonate (DEPC) water at a mass ratio of 1:1. The absorbance at 260 nm is measured using a Nanodrop ultra-micro spectrophotometer to prepare an antisense oligonucleotide solution with an OD of 0.4. According to the mass ratio of antisense oligonucleotide to dextran nanosilica particles of 1:200, the dextran nanosilica particle solution of Example 2 is added. After mixing evenly, it is incubated at 4 °C for 12 h to obtain a crude product solution;

[0263] (2) The crude product solution is centrifuged (5000×g * 15 min) through a Nanosep centrifugal device (M W cutoff, 10 kDa, Millipore) and eluted 5 times with PBS to prepare 100 μL of nanocomposite solution.

[0264] Comparative Example 2 Preparation of 3D printed microneedles

[0265] The preparation of 3D printed microneedles comprises the following steps:

[0266] (1) The methacrylated polylysine / methacrylated sodium hyaluronate solution is injected into the polydimethylsiloxane negative mold. After vacuum degassing (-0.1 MPa, 2 min) treatment, it is dried at room temperature for 2 h, and the remaining solution and bubbles are scraped off to prepare 10 μL of the needle tip layer;

[0267] (2) The polyethylene glycol diacrylate solution is injected into the polydimethylsiloxane negative mold. After vacuum degassing (-0.1 MPa, 2 min), it is ultraviolet cured (λ = 405 m, light intensity 25 mW·cm -2 , 5 cm away from the mold surface) for 90 s and then cooled to room temperature to prepare 500 μL of the base layer;

[0268] (3) After standing for 1 - 2 h, demolding is carried out to obtain 3D printed microneedles (H / P-MN) with a double-layer structure.

[0269] Test Example 1 Study on the stability of the nanocomposite of the present invention

[0270] Taking the nanocomposite of Example 5 as an example, it is dispersed in deionized water and PBS buffer (pH 7.4) and stored at 4 °C for 7 days, and the change in the hydrated particle size is dynamically monitored. The results are shown in Figure 2 a and Figure 2b.

[0271] This nano - composite has excellent stability in physiological environment.

[0272] Test Example 2 Safety investigation of the nano - composite of the present invention

[0273] Taking the nano - composite of Example 5 as an example, the growth inhibitory activity of the nano - composite on L929 cells was detected by the Cell Counting Kit - 8 (CCK - 8) method.

[0274] The diluted cells were inoculated into a sterile 96 - well plate, 100 μL per well (i.e., the number of cells per well was 10 4 cells). It was cultured in a cell incubator at 37 °C for 24 h. Then 100 μL of sample solutions with different concentrations were added. α - DMEM with 10% fetal bovine serum was set as the blank control, cell culture medium as the negative control, and 50% dimethyl sulfoxide (DMSO) as the positive control, and they were cultured in a cell incubator at 37 °C for 24 h. After adding 10 μL of CCK - 8 solution, the samples were cultured in a cell incubator at 37 °C for 1 h. The absorbance values of each sample well were detected using a multi - functional microplate reader at a wavelength of 450 nm, and the cell survival rate was calculated.

[0275] Cell survival rate = ((ODsample - ODnegative control) / (ODnegative control - ODblank))×100%

[0276] The results are shown in Figure 3 a. The survival rate of L929 cells reached 94.6 ± 3.8% during 48 - h culture with the nano - composite, and it had no significant effect on cell activity.

[0277] The hemolysis rate of the nano - composite was 2.8 ± 0.3%, and the red blood cells remained intact biconcave disc - shaped structures after being treated with the nano - composite, without rupture or shrinkage, indicating good blood compatibility. The results are shown in Figure 3 b - Figure 3 c. The nano - composite of the present invention has excellent safety.

[0278] Test Example 3 Study on the antibacterial activity of the nano - composite of the present invention

[0279] The cryopreserved Escherichia coli and Staphylococcus aureus were inoculated into LB liquid medium and cultured with shaking at 37 °C and 180 rpm until the logarithmic growth phase (OD 600 = 0.5) to prepare a bacterial suspension. After centrifugation, the concentration of the bacterial solution was adjusted to 1×10 7 CFU·mL -1, Take 100 μL and add it to a 96-well plate. Then, add 100 μL of the ASO solution (mix Ec acpP and Sau ftsZ at a mass ratio of 1:1, dissolve in DEPC water, and prepare an ASO solution with an OD of 0.4), the nano-complex of Comparative Example 1, and the nano-complex of Example 5 (nucleic acid concentration 5 μM) solution. Use LB medium as the blank (Control group), and set up 3 replicate wells. Incubate continuously in a constant temperature shaker (37 °C, 180 rpm) for 24 h, and measure the OD 600 value every 3 h to plot the bacterial growth curve.

[0280] The results are shown in Figure 4 a and Figure 4 b. The nano-complex of the present invention has excellent antibacterial activity.

[0281] Test Example 4 Study on the bacterial uptake of the nano-complex of the present invention

[0282] Flow cytometry was used to analyze the uptake rates of Escherichia coli and Staphylococcus aureus for the nano-complex (ASO-Cy5) of Example 10. The ASO-Cy5 solution was used as the negative control, and Lipofectamine 3000 (Lipo3000) encapsulating ASO-Cy5 was used as the positive control.

[0283] (1) Live / dead staining: Take 1.0×10 9 CFU of bacterial suspension, add 1 μL of SYTO 9 (3.34 mM, Thermo Fisher) and 1 μL of propidium iodide (PI, 1.5 mM, Thermo Fisher), and incubate in the dark for 15 min. SYTO 9 labels the intact cells of live bacteria (green fluorescence, Ex / Em: 480 / 500 nm), and PI penetrates the damaged membranes of dead bacteria (red fluorescence, Ex / Em: 535 / 617 nm);

[0284] (2) Detection of ASO-Cy5 signal: Use a flow cytometer to directly detect the Cy5-labeled nano-complex (far red fluorescence, Ex / Em: 640 / 670 nm). Set the threshold to exclude debris signals. Collect 10,000 bacterial events for each sample. FlowJo software (v7.6.1) analyzes the bacterial population by gating with FSC / SSC, and calculates the percentage of Cy5 + in live cells + cell percentage.

[0285] The results are shown in Figure 5 . The uptake rates of wild-type Escherichia coli ( Figure 5 a) and Staphylococcus aureus ( Figure 5 b) for the nano-complex of the present invention are 46.4% and 37.1% respectively.

[0286] Test Example 5 Investigation on the Bacterial mRNA Degradation Mediated by the Nanocomplex of the Present Invention

[0287] Using 16S rRNA as an internal reference, SYBR Green method was used for qRT-PCR detection to investigate the degradation effect of the nanocomplex of the present invention on the transcriptional mRNA of the acpP gene of Escherichia coli and the transcriptional mRNA content of the fmhB gene of Staphylococcus aureus.

[0288] PBS (negative control), the nanocomplex of Comparative Example 1 (5 μM), and the nanocomplexes of Example 5 (1 μM, 2 μM, and 5 μM) were incubated with 5.0×10 9 CFU of Escherichia coli and 5.0×10 9 CFU of Staphylococcus aureus at 37°C and 500 rpm for 3 h, respectively. Then, the total RNA of bacteria was extracted by Trizol method, and the eluted RNA was stored at -80°C.

[0289] RNA reverse transcription experiment: First, remove gDNA and then perform reverse transcription. When removing gDNA, prepare reaction solution of System 1 (10 μL) or System 2 (16 μL) on ice, react at 42°C for 2 min, and then store at 4°C; for reverse transcription, prepare the reaction solution on ice, place it in a PCR instrument, react at 37°C for 15 min and at 85°C for 5 s in sequence, and then store at 4°C.

[0290] Real-time quantitative PCR: In a 20 μL reaction system of FastStart Universal SYBR Green Master, it contains 10 μL of 2x SuperReal PreMix Plus, 0.6 μL of forward primer (10 μM), 0.6 μL of reverse primer (10 μM), appropriate cDNA template and enzyme-free water (total volume is 20 μL). React in a fluorescence quantitative PCR instrument. The results are shown in Figure 6 .

[0291] The nanocomplex of the present invention mediated the inhibition of the mRNA level of the acpP gene in Escherichia coli to (4.0 ± 1.0)% (p < 0.01), and there was a significant difference compared with the expression level of the acpP gene in the control group (89.0 ± 15.0)% (p < 0.01).

[0292] The nanocomplex of the present invention mediated the inhibition of the mRNA level of the ftsZ gene in Staphylococcus aureus to (26.0 ± 3.0)%, and there was a significant difference compared with the expression level of the ftsZ gene in the control group (81.0 ± 19.0)% (p < 0.01).

[0293] Test Example 6Performance Characterization of 3D Printed Microneedles of the Present Invention

[0294] The morphology of the 3D printed microneedles of Example 11 was characterized by a digital microscope, and the results are shown in Figure 7 a and Figure 7 b. The microneedle array presents a complete mushroom bionic structure, the demolding integrity rate of the needle body is ≥90%, and the curvature radii of the needle tip and the needle body are consistent with the 3D printing template design values.

[0295] Test Example 7 Investigation on the Mechanical Properties of 3D Printed Microneedles of the Present Invention

[0296] The mechanical properties of the 3D printed microneedles of the present invention were detected by an MTS C42.502y texture analyzer (MTS Systems, USA). The 3D printed microneedle patch of Example 11 was fixed on a metal table, and the needle tip was vertically aligned with a Φ13.3 mm cylindrical probe, and a vertical pressure was applied at a rate of 0.05 mm·min -1 The initial spacing was 0.5 cm, the trigger force was 0.05 N, and the termination force was 70 N. The following parameters were calculated through the force-displacement curve: Fracture Force: the maximum load when the microneedle undergoes structural damage (target value: ≥0.030 N / needle), and the results are shown in Figure 8 . The fracture force of the 3D printed microneedles of the present invention is (0.28 ± 0.03 N / needle).

[0297] Test Example 8 Investigation on the In Vitro Drug Release of 3D Printed Microneedles of the Present Invention

[0298] The release of the nano-complex in the 3D printed microneedles of Example 11 was detected using a Franz diffusion cell (effective diffusion area 1.77 cm 2 , receiving cell volume 7 mL).

[0299] The receiving phase was degassed PBS (pH 7.4). Under the conditions of stirring (200 ± 5 rpm) and 32 ± 0.5 °C, 500 μL of the PBS receiving solution was accurately collected by an automatic sampling system at 1 min, 5 min, 15 min, 30 min, 60 min, and 180 min respectively (synchronous replenishment of liquid to maintain osmotic pressure balance). After the samples were purified by a 0.22 μm PVDF filter membrane, they were immediately transferred to a pre-cooled brown glass bottle (stored in the dark at 4 °C). The concentration of the nano-complex was quantitatively analyzed by ultraviolet-visible spectrophotometry (UV-2700, Shimadzu, λ = 260 nm), and the cumulative release rate was calculated by the standard curve method (R 2 = 0.9992). The results are shown in Figure 9 .

[0300] The baseline drift was corrected daily, and a blank microneedle control was set for each experiment. All data were analyzed by one-way ANOVA using SPSS 26.0 (α = 0.05).

[0301] Test Example 9 Study on the in vivo antibacterial and wound healing promoting properties of the 3D printed microneedles of the present invention

[0302] Twenty-four male SD rats aged 6 - 8 weeks with a body weight of 180 - 200 g were divided into 4 groups, with 6 rats in each group.

[0303] After the rats were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital solution, the back hair was shaved. The remaining hair was removed using depilatory cream. After the skin was disinfected with iodophor, four circular full-thickness skin defect wound models with a diameter of 1.5 cm were established on the back of the rats using surgical scissors. The surgical modeling was on day 0, and drugs were administered for 2 days on the 1st and 2nd days after modeling:

[0304] (1) Uninfected group (Control): After establishing the circular full-thickness skin defect wound model, the wound was not treated;

[0305] (2) Infected group (Infected): After establishing the circular full-thickness skin defect wound model, 100 μL of bacterial suspension containing 1×10 7 CFU (the ratio of the number of Escherichia coli to Staphylococcus aureus in the bacterial suspension was 1:1) was dropped onto the surface of the wound, and the wound was not treated;

[0306] (3) Control group (H / P-MN): After establishing the circular full-thickness skin defect wound model, 100 μL of bacterial suspension containing 1×10 7 CFU (the ratio of the number of Escherichia coli to Staphylococcus aureus in the bacterial suspension was 1:1) was dropped onto the surface of the wound, and then the microneedles of Comparative Example 2 were inserted into the surface of the wound (administered 2 times a day, 1 microneedle each time);

[0307] (4) Treatment group (MNDS): After establishing the circular full-thickness skin defect wound model, 100 μL of bacterial suspension containing 1×10 7 CFU (the ratio of the number of Escherichia coli to Staphylococcus aureus in the bacterial suspension was 1:1) was dropped onto the surface of the wound, and then the microneedles of Example 11 were inserted into the surface of the wound (administered 2 times a day, 1 microneedle each time).

[0308] Two days after drug administration, bacteria at the wound sites of the Control group, Infected group, H / P-MN group, and MNDS group were dipped and plated, and then placed in an incubator at 37°C for 24 h. The bacterial culture plates were taken out, colony counting was performed, and photos were taken to compare the bacterial numbers of wounds in different groups. The results are shown in Figure 10a, the CFU counts in the H / P-MN group and the MNDS treatment group were significantly lower than those in the Infected group, and the CFU count in the H / P-MN group was about 2-3 times that of the MNDS treatment group.

[0309] As Figure 10b shown, compared with the Infected group, there were significant differences in the wound healing rates of the H / P-MN group and the MNDS group on the 5th day of drug administration (P<0.05). Compared with the Infected group, there were significant differences in the wound healing rates of the H / P-MN group and the MNDS group on the 7th day of drug administration (P<0.05).

[0310] On the 2nd day, bacteria were swabbed from the wound surface and spread on an agar plate for colony counting. During the observation of the wound surface, obvious exudate appeared in the Infected group and a bacterial biofilm was formed on the 2nd day. No obvious bacterial biofilm was formed in the H / P-MN group and the MNDS group until the 13th day. There was only a small amount of exudate on the wound surface, and no significant pathological changes were observed in the main organs of the rats in the MNDS group.

[0311] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or deformations according to the present invention, as long as they do not depart from the spirit of the present invention, they shall fall within the scope of the present invention.

Claims

1. A 3D printed antibacterial microneedle, wherein the microneedle is a bionic mushroom-like structure made of a drug-loaded tip layer, a needle body layer, a drug-loaded reservoir layer, and a base layer. Among them, The volume ratio of the drug-loaded tip layer: the needle body layer: the drug-loaded reservoir layer: the base layer is 1:1:20 - 50:20 - 50; The drug-loaded tip layer is prepared by loading a nano-complex with soluble polyvinylpyrrolidone. The mass-volume ratio of soluble polyvinylpyrrolidone: nano-complex solution in the drug-loaded tip layer is 100 - 200:1 (mg / mL); The needle body layer is prepared by photo-crosslinking after concentrating methacrylated polylysine and methacrylated sodium hyaluronate. The mass ratio of methacrylated polylysine: methacrylated sodium hyaluronate in the needle body layer is 1:0.25 - 1; The drug-loaded reservoir layer is prepared by loading a nano-complex with soluble polyvinylpyrrolidone. The mass-volume ratio of soluble polyvinylpyrrolidone: nano-complex solution in the drug-loaded reservoir layer is 100 - 200:1 (mg / mL); The base layer is made by photo-crosslinking of non-degradable polyethylene glycol diacrylate.

2. The microneedle according to claim 1, wherein the drug-loaded reservoir layer is a circular patch.

3. The microneedle according to any one of claims 1 - 2, wherein the base layer is a square patch.

4. The microneedles according to any one of claims 1-3, wherein the nano-complex is made of an antisense oligonucleotide and dextran nanosilica particles in a mass ratio of 1:100-300, wherein, The antisense oligonucleotide is selected from any one or a combination of a nucleotide sequence against Gram-positive bacteria and a nucleotide sequence against Gram-negative bacteria. The dextran nanosilica particles are made by covalently binding dextran to the surface of nanosilica particles. The dextran is selected from any one or a combination of maltodextrin and diethylaminoethyl dextran.

5. The preparation method of the 3D printed antibacterial microneedles according to any one of claims 1-4, wherein the microneedles are biomimetic mushroom-shaped structures made of a drug-loaded tip layer, a needle body layer, a drug-loaded reservoir layer, and a base layer. Among them, The volume ratio of the drug-loaded tip layer: the needle body layer: the drug-loaded reservoir layer: the base layer is 1:1:20 - 50:20 - 50. The preparation method of the microneedle comprises the following steps: (1) The drug-loaded tip layer is prepared by loading a nano-complex with soluble polyvinylpyrrolidone. The mass-volume ratio of soluble polyvinylpyrrolidone: nano-complex solution in the drug-loaded tip layer is 100 - 200:1 (mg / mL); (2) The needle body layer is prepared by photo-crosslinking methacrylated polylysine and methacrylated sodium hyaluronate. The mass ratio of methacrylated polylysine: methacrylated sodium hyaluronate in the needle body layer is 1:0.25 - 1; (3) The drug-loaded reservoir layer is prepared by loading a nano-complex with soluble polyvinylpyrrolidone. The mass-volume ratio of soluble polyvinylpyrrolidone: nano-complex solution in the drug-loaded reservoir layer is 100 - 200:1 (mg / mL); (4) The base layer is made by photo-crosslinking of non-degradable polyethylene glycol diacrylate.

6. The method according to claim 5, wherein the preparation of the drug-loaded tip layer comprises the following steps. Under stirring (100 - 200 rpm), in the required amount of nano-complex solution, polyvinylpyrrolidone is added according to the mass-volume ratio of polyvinylpyrrolidone: nano-complex solution of 100 - 200:1 (mg / mL). The mixture solution made of polyvinylpyrrolidone and nano-complex solution is injected into a polydimethylsiloxane negative mold. After vacuum degassing, it is dried for 1 - 2 h to obtain.

7. The method according to any one of claims 5-6, wherein the preparation of the needle body layer comprises the following steps: under stirring (100-200 rpm), mix methacrylated polylysine and sodium hyaluronate methacrylate in a mass ratio of 1:0.25-1, then make a 5% solution with PBS (pH 7.4), add lithium phenyl-2,4,6-trimethylbenzoylphosphinate with a concentration of 0.5%, mix for 1-2 h, filter through a 0.22 μm filter membrane, then inject it into a polydimethylsiloxane negative mold, degas under vacuum, and then cure by ultraviolet light and dry for 1-2 h to obtain the product.

8. The method according to any one of claims 5-7, wherein the preparation of the drug-loaded reservoir layer comprises the following steps: under stirring (100-200 rpm), add polyvinylpyrrolidone to the required amount of the nano-complex solution at a mass-to-volume ratio of 100-200:1 (mg / mL), inject the mixture solution of polyvinylpyrrolidone and the nano-complex solution into a polydimethylsiloxane negative mold, degas under vacuum, and then dry for 8-10 h to obtain the product.

9. The method according to any one of claims 5-8, wherein the preparation of the base layer comprises the following steps: under stirring (100-200 rpm), mix polyethylene glycol diacrylate (PEGDA400) and polyethylene glycol diacrylate (PEGDA600) in a mass ratio of 1:1-5, add 2,4,6-trimethylbenzoyl-bis(p-tolyl)phosphine oxide with a weight percentage concentration of 0.5%, mix for 0.5-1 h, inject the prepared polyethylene glycol diacrylate solution into a polydimethylsiloxane negative mold, and then cure by ultraviolet light to obtain the product.

10. The application of the 3D printed antibacterial microneedle according to any one of claims 1-4 or the 3D printed antibacterial microneedle prepared according to any one of claims 5-9 for antibacterial treatment.

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